Summary Histone Deacetylase (HDAC) proteins are epigenetic modulators that have a clear and well studied role in gene expression regulation. HDAC proteins influence gene expression by deacetylating acetyl-lysine residues on nucleosomal histone protein substrates. Recently a wide variety of acetylated proteins have been discovered, which suggests that HDAC proteins likely deacetylate substrates in addition to histones. Through non-histone substrates, HDAC proteins may play an expanded role in cellular processes outside of gene expression, including cell signaling and communication. Unfortunately, identification of non- histone HDAC substrates has been largely serendipitous because facile methods to systematically discover in cellulo targets of HDAC deacetylation are lacking. This application outlines development of ?substrate trapping? mutants of HDAC proteins for substrate discovery. In prior work, we created roughly 70 inactive HDAC1 mutants that have the potential to stably bind substrates and facilitate their purification and identification. We present here exciting preliminary results identifying and validating several novel HDAC1 substrates using the trapping strategy. Our further goals in this application are to screen additional mutants for optimal substrate binding properties, with subsequent identification of substrates of HDAC1 in mammalian cells (Specific Aim 1). Given the high sequence similarity within the HDAC family, we will extend these studies to HDAC6 because its cytoplasmic localization suggests a non-gene regulatory function (Specific Aim 2). Finally, to more clearly distinguish active site binding substrates from associated proteins in the trapping experiments, we will generate HDAC1-selective inhibitors for use as competitive control compounds (Specific Aim 3). In total, the significant outcome of this application is the creation of a trapping mutant strategy for the unbiased identification of HDAC substrates. With the availability of the first systematic tool to identify substrates, our long-term goal is to apply the technology to decipher the substrate specificity of HDAC proteins in cell biology. Given the role of HDAC proteins in epigenetics, and their possible role in cell signaling, substrate trapping mutants have the potential to augment our understanding of cell biology and embolden drug design efforts targeting HDAC proteins.